Abstract: To isolate and prepare the biflavonoids reference substance with high-purity from Ginkgo biloba L.,then investigate their antioxidant activity.High-purity biflavonoids were prepared by 60% ethanol-water extract of Ginkgo biloba L,through antisolvent precipitation and semi-preparative HPLC.Crude biflavonoids (purity:80.40%) was obtained by antisolvent precipitation process,then purified by semi-preparative HPLC with the mobile phase comprising of methanol-water in a gradient elution manner.Under the optimal preparation conditions,four biflavonoids were obtained and identified as amentoflavone (98.45%),bilobetin (98.66%)，ginkgetin isomers (98.87%) and sciadopitysin (99.29%) with the yield of 22.5,23.8,192.5,71.8 (mg/kg) respectively,which met the requirements of TCM reference standards,and examined their antioxidant activity,then completely separate the ginkgetin isomers by the use of 60% acetonitrile and organic acid aqueous solution.The preparation method is rapid and simple,and the obtained biflavonoids have high purity and high yield.This study provides important technical support for the research and development of one-component ginkgo biflavonoids.

Key words: biflavonoids, antisolvent precipitation, TCM reference substance, semi-preparative HPLC, Ginkgo biloba leaves